The present invention relates to a Neurturin (xe2x80x9cNTNxe2x80x9d) receptor designated NTNRxcex1, and provides for NTNRxcex1-encoding nucleic acid and amino acid sequences. In particular, the invention relates to native sequence NTNRxcex1, NTNRxcex1 variants, soluble NTNRxcex1 variants including NTNRxcex1 extracellular domain, chimeric NTNRxcex1, and antibodies which bind to the NTNRxcex1 (including agonist and neutralizing antibodies), as well as various uses for these molecules. It also relates to assay systems for detecting ligands to NTNRxcex1, systems for studying the physiological role of NTN, diagnostic techniques for identifying NTN-related conditions, therapeutic techniques for the treatment of NTN-related and NTNRxcex1-related conditions, and methods for identifying molecules homologous to NTNRxcex1.
Neurotrophic factors such as insulin-like growth factors, nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, -4/5 and -6, ciliary neurotrophic factor, GDNF, and neurturin have been proposed as potential means for enhancing specific neuronal cell survival, for example, as a treatment for neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer""s disease, stroke, epilepsy, Huntington""s disease, Parkinson""s disease, and peripheral neuropathy. It would be desirable to provide additional therapy for this purpose. Protein neurotrophic factors, or neurotrophins, which influence growth and development of the vertebrate nervous system, are believed to play an important role in promoting the differentiation, survival, and function of diverse groups of neurons in the brain and periphery. Neurotrophic factors are believed to have important signaling functions in neural tissues, based in part upon the precedent established with nerve growth factor (NGF). NGF supports the survival of sympathetic, sensory, and basal forebrain neurons both in vitro and in vivo. Administration of exogenous NGF rescues neurons from cell death during development. Conversely, removal or sequestration of endogenous NGF by administration of anti-NGF antibodies promotes such cell death (Heumann, J. Exp. Biol., 132:133-150 (1987); Hefti, J. Neurosci., 6:2155-2162 (1986); Thoenen et al., Annu. Rev. Physiol., 60:286-335 (1980)).
Additional neurotrophic factors related to NGF have since been identified. These include brain-derived neurotrophic factor (BDNF) (Leibrock, et al., Nature, 341:149-152 (1989)), neurotrophin-3 (NT-3) (Kaisho, et al., FEBS Lett., 266:187 (1990); Maisonpierre, et al., Science, 247:1446 (1990); Rosenthal, et al., Neuron, 4:767 (1990)), and neurotrophin 4/5 (NT-4/5) (Berkmeier, et al., Neuron, 7:857-866 (1991)).
Neurotrophins, similar to other polypeptide growth factors, affect their target cells through interactions with cell surface receptors. According to current understanding, two kinds of transmembrane glycoproteins act as receptors for the known neurotrophins. Equilibrium binding studies have shown that neurotrophin-responsive neuronal cells possess a common low molecular weight (65,000-80,000 Daltons), a low affinity receptor typically referred to as p75LNGFR or p75, and a high molecular weight (130,000-150,000 Dalton) receptor. The high affinity receptors are members of the trk family of receptor tyrosine kinases.
Receptor tyrosine kinases are known to serve as receptors for a variety of protein factors that promote cellular proliferation, differentiation, and survival. In addition to the trk receptors, examples of other receptor tyrosine kinases include the receptors for epidermal growth factor (EGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF). Typically, these receptors span the cell membrane, with one portion of the receptor being intracellular and in contact with the cytoplasm, and another portion of the receptor being extracellular. Binding of a ligand to the extracellular portion of the receptor induces tyrosine kinase activity in the intracellular portion of the receptor, with ensuing phosphorylation of various intracellular proteins involved in cellular signaling pathways.
Glial cell line-derived neurotrophic factor (xe2x80x9cGDNFxe2x80x9d) and Neurturin (xe2x80x9cNTNxe2x80x9d) are two, recently identified, structurally related, potent survival factors for sympathetic sensory and central nervous system neurons (Lin et al. Science 260:1130-1132 (1993); Henderson et al. Science 266:1062-1064 (1994); Buj-Bello et al., Neuron 15:821-828 (1995); Kotzbauer et al. Nature 384:467-470 (1996)). Recently, GDNF was shown to mediate its actions through a multi-component receptor system composed of a ligand binding glycosyl-phosphatidyl inositol (GPI) linked protein (designated GDNFRxcex1; also designated GFR-alpha-1) and the transmembrane receptor tyrosine kinase Ret (Treanor et al. Nature 382:80-83 (1996); Jing et al. Cell 85:1113-1124 (1996); Trupp et al. Nature 381:785-789 (1996); Durbec et al. Nature 381:789-793 (1996)). The mechanism by which the NTN signal is transmitted has not been elucidated.
Aberrant expression of receptor tyrosine kinases (xe2x80x9cRTKxe2x80x9d) correlates with transforming ability. For example, carcinomas of the liver, lung, breast and colon show elevated expression of Eph RTK. Unlike many other tyrosine kinases, this elevated expression can occur in the absence of gene amplification or rearrangement. Moreover, Hek, a human RTK, has been identified as a leukemia-specific marker present on the surface of a pre-B cell leukemia cell line. As with Eph, Hek also was overexpressed in the absence of gene amplification or rearrangements in, for example, hemopoietic tumors and lymphoid tumor cell lines. Over-expression of Myk-1 (a murine homolog of human Htk (Bennett et al., J. Biol. Chem., 269(19):14211-8 (1994)) was found in the undifferentiated and invasive mamnmary tumors of transgenic mice expressing the Ha-ras oncogene. (Andres et al., Oncogene, 9(5):1461-7 (1994) and Andres et al., Oncogene, 9(8):2431 (1994)). Ret, the product of the c-ret proto-oncogene, is a member of the receptor tyrosine kinase superfamily.
In addition to their roles in carcinogenesis, a number of transmembrane tyrosine kinases have been reported to play key roles during development. Some receptor tyrosine kinases are developmentally regulated and predominantly expressed in embryonic tissues. Examples include Cek1, which belongs to the FGF subclass, and the Cek4 and Cek5 tyrosine kinases (Pasquale et al., Proc. Natl. Acad Sci., USA, 86:5449-5453 (1989); Sajjadi et al., New Biol., 3(8):769-78 (1991); and Pasquale, Cell Regulation, 2:523-534(1991)). Eph family members are expressed in many different adult tissues, with several family members expressed in the nervous system or specifically in neurons (Maisonpierre et al., Oncogene, 8:3277-3288 (1993); Lai et al., Neuron, 6:691-704 (1991)).
The aberrant expression or uncontrolled regulation of any one of these receptor tyrosine kinases can result in different malignancies and pathological disorders. Therefore, there exists a need to identify means to regulate, control and manipulate receptor tyrosine kinases (xe2x80x9cRTKxe2x80x9d) and their associated ligands or GPI-linked receptors, in order to provide new and additional means for the diagnosis and therapy of receptor tyrosine kinase pathway-related disorders and cellular processes. The present application provides the clinician and researcher with such means by providing new molecules that are specific for interacting with certain RTK receptors. These compounds and their methods of use, as provided herein, allow exquisite therapeutic control and specificity. Accordingly, it is an object of the present invention to provide an improved therapy for the prevention and/or treatment of neurological conditions and other conditions in which certain neurotrophic signaling pathways play a role.
These and other objects of the invention will be apparent to the ordinarily skilled artisan upon consideration of the specification as a whole.
A NTN receptor termed NTNRxcex1, a soluble form of the receptor, and a NTNRxcex1 extracellular domain (xe2x80x9cECDxe2x80x9d) are disclosed herein. Also disclosed are NTNRxcex1 polypeptides, optionally conjugated with or fused to molecules which increase the serum half-lives thereof, and optionally formulated as pharmaceutical compositions with a physiologically acceptable carrier.
Soluble NTNRxcex1, including chimeric NTNRxcex1 molecules such as NTNRxcex1 ECD immunoadhesins (having long serum half-lives) and epitope-tagged NTNRxcex1 ECD, that retain both ligand binding, preferably NTN binding, and receptor signaling function (via Ret receptor tyrosine kinase) can be used to impart, restore, or enhance NTNRxcex1-ligand (preferably NTN) responsiveness to cells. This responsiveness includes ligand-binding, Ret tyrosine phosphorylation and Ret-mediated downstream activity, which can result in modulation of cell activity such as survival or growth. The embodiments find use in vivo, in vitro or ex vivo. Soluble NTNRxcex1 forms that bind NTN but fail to interact with and activate Ret can be used as an antagonist to NTN ligand (by binding and sequestering NTN) to reduce activation of endogenous NTNRxcex1. This is useful in conditions characterized by excess levels of NTN ligand and/or excess NTNRxcex1 activation in a mammal. Bispecific immunoadhesins (for example, combining a NTNRxcex1-ligand binding activity with a ligand-binding domain of another cytokine or neurotrophic factor receptor) can form high affinity binding complexes for NTNRxcex1-ligands and another factors, providing either antagonist activity (when Ret-activating function is absent) or a means to enhance the attached ligands delivery.
Pharmaceutical compositions of soluble NTNRxcex1, preferably ECD, can optionally further include an NTNRxcex1 ligand, preferably NTN. Such compositions are useful where it is desirable to prolong the half-life of the ligand, provide slow, sustained release of ligand, impart NTNRxcex1-ligand responsiveness to a target cell, and/or activate or enhance endogenous cellular NTNRxcex1 or Ret activity directly. Optionally, the composition further contains one or more cytokines, neurotrophic factors, or their agonist antibodies.
Also provided are methods for identifying a molecule which binds to and/or activates NTNRxcex1. Thus assays are provided to screen for or identify NTNRxcex1-ligand molecules (such as peptides, antibodies, and small molecules) that are agonists or antagonists of NTNRxcex1. Such methods generally involve exposing an immobilized NTNRxcex1 to a molecule suspected of binding thereto and determining binding of the molecule to the immobilized NTNRxcex1 and/or evaluating whether or not the molecule activates (or blocks activation of) the NTNRxcex1. In order to identify such NTN ligands, the NTNRxcex1 can be expressed on the surface of a cell and used to screen libraries of synthetic candidate compounds or naturally-occurring compounds (e.g., from endogenous sources such as serum or cells). NTNRxcex1 can also be used as a diagnostic tool for measuring serum levels of endogenous or exogenous NTNRxcex1-ligand.
In a further embodiment, a method for purifying an NTNRxcex1-ligand is provided. This finds use in commercial production and purification of therapeutically active molecules that bind to this receptor. In one embodiment the molecule of interest (generally in a composition comprising one or more contaminants) is adsorbed to immobilized NTNRxcex1 (e.g., NTNRxcex1 immunoadhesin immobilized on a protein A resin). The contaminants, by virtue of their inability to bind to the NTNRxcex1, will generally not bind the resin. Accordingly, it is then possible to recover the molecule of interest from the resin by changing the elution conditions, such that the ligand molecule is released from the immobilized receptor.
Antibodies are provided that specifically bind to NTNRxcex1. Preferred antibodies are monoclonal antibodies that are non-immunogenic in a human and bind to an epitope in the extracellular domain of the receptor. Preferred antibodies bind the NTNRxcex1 with an affinity of at least about 106 L/mole, more preferably 107 L/mole. Preferred antibodies are agonist antibodies.
Antibodies, which bind to NTNRxcex1, can be optionally fused to a heterologous polypeptide. The antibody or fusion finds particular use to isolate and purify NTNRxcex1 from a source of the receptor.
In a further aspect is provided a method for detecting NTNRxcex1 in vitro or in vivo which includes the steps of contacting an NTNRxcex1 antibody with a sample suspected of containing the receptor, and detecting if binding has occurred.
For certain applications it is desirable to have an agonist antibody. Such agonist antibodies are useful for activating NTNRxcex1 as described for NTNRxcex1-ligands such as NTN. Furthermore, these antibodies are useful to treat conditions in which an effective amount of NTNRxcex1 activation leads to a therapeutic benefit in the mammal. For example, the agonist antibody can be used to elicit an NTN response in a cell comprising NTNRxcex1 and, preferably, Ret. For therapeutic applications it is desirable to prepare a composition having the agonist antibody and a physiologically acceptable carrier. Optionally, the composition further contains one or more cytokines, neurotrophic factors, or their agonist antibodies.
In other embodiments, the antibody is a neutralizing antibody. Such molecules can be used to treat conditions characterized by unwanted or excessive activation of NTNRxcex1.
In addition to the above, the invention provides isolated nucleic acid molecules, expression vectors and host cells encoding NTNRxcex1 which can be used in the recombinant production of NTNRxcex1 as described herein. The isolated nucleic acid molecules and vectors are also useful to prepare transgenic animals, for gene therapy applications to treat patients with NTNRxcex1 defects or increase responsiveness of cells to NTNRxcex1 ligands, or alternatively to decrease NTNRxcex1 activity (as by use of antisense nucleic acid).